PROJECT ABSTRACT Idiopathic pulmonary fibrosis (IPF) of the distal lung is characterized by spatially heterogeneous areas of fibroblasts/myofibroblasts and accumulation of excess extracellular matrix (ECM) that disrupts the alveolar architecture. Alveolar epithelial cells are now thought to directly contribute to the development and progression of fibrosis, but how new ECM deposition impacts alveolar epithelia function and feedbacks to reinforce fibrotic remodeling is unknown. This is due in part to the lack of tools to study ECM dynamics or to directly measure cell fate in response to altered ECM in vivo. Organoid cultures can generate an in vivo-like complement of alveolar tissue; however, current in vitro models depend on the use of Matrigel matrices, which feature variable compositions and is not conducive to controlled manipulations. The overall focus of the proposed work is to connect changes in secreted alveolar ECM cues and epithelial cell function (K99 phase) and epithelia to mesenchyme signaling (R00). During the K99 phase, defined hydrogel matrices will be developed to form alveolar organoids in a defined microenvironment and determine how newly secreted ECM composition and mechanics guide epithelial cell function. Using this platform, the secreted ECM will be externally crosslinked to examine whether ECM stiffening promotes epithelial cells dysfunction. With an understanding of how secreted ECM initiates epithelial cell dysfunction, the second aim will determine how the secreted ECM alters the response of epithelial cells to signals from the mesenchyme during the R00 phase. We will determine if the accumulation of ECM changes the interaction between AT2 cells and mesenchymal cells, and whether this reinforces fibrotic remodeling. Next, microstructured hydrogels will be used to control for spatial relationships and examine the effect of the physical separation of epithelial and mesenchymal cells on epithelial cell function. To understand the bidirectional signaling of cells and their continuously changing surroundings within the alveolar niche, this research will use organoid cultures and engineering approaches to manipulate and deconstruct cell-ECM interactions. The outcomes will comprise identification of new ECM mediated mechanisms involved in alveolarization and reparative processes, and provide new avenues for testing therapeutics modulating paracrine signaling pathways involved in IPF. Importantly, this proposal comprises a rigorous training plan that will build the foundation to advance the applicant?s career in biomedical research. Specifically, the K99 training will consist of learning mouse models, lineage tracing, and primary cell isolation to establish the foundation towards investigating cell-cell signaling using engineered hydrogels during the independent investigator R00 phase.